Understanding in greater molecular detail the key role played by receptor-mediated endocytosis in a variety of cellular process, such as lipid metabolism and its derangements (e.g., familian hypercholesterolemia) and peptide hormone metabolism and its derangements (e.g., some forms of diabetes) is the long-term objective of the proposed research. Receptor-mediated endocytosis is a process by which cells bind and internalize transport proteins and peptide hormones. The specific aims of this research are 1) to isolate and characterize Chinese hamster ovary (CHO) cells carrying mutations that affect receptor-mediated endocytosis of low density lipoprotein (LDL), 1) to determine the molecular basis for the genetic defects in the mutant CHO cells, 3) to extend the genetic and biochemical analysis of receptor-mediated endocytosis of LDL to the receptor-mediated endocytosis of other receptor-ligands, e.g., mannose-6-phosphate derivatives, and 4) to investigate the feasability of extending these stdies to other types of permanent cell lines (e.g., 3T3, L, NRK) in culture. To permit the isolation of these mutants, I have developed a technique for reconstituting LDL so that it can kill cells when it is processed through the LDL receptor-mediated endocytic pathway. This toxic LDL has been successfully used to isolate mutants which do not express LDL receptor binding activity. Previously isolated mutants which fall into two complementation groups will permit the isolation and biochemical characterization of an heretofore unrecognized factor which is required for the expression of LDL receptor activity. To study the receptor-mediated endocytosis of mannose-6-phosphate derivatives, we will crosslink these molecules to reconstituted LDL and mutants will be isolated and analyzed. To isolate mutants with defects affecting both receptor systems, we will use a mixture of the toxic LDL and the toxid LDL crosslinked to a mannose-6-phosphate derivative.